ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-9 



Gram stain. The Gram stain was until recently an entirely empiri- 

 cal procedure for distinguishing between two groups of organisms, the 

 actual significance of which was not understood. Since 1940, how- 

 over, the work of Henry and Stacey (194'3), of Bartholomew and Um- 

 breit (1944) and others has shown that a positive reaction is de- 

 l)endent upon the presence of ribonucleic acid in the outer layers of 

 the cells, which can be removed by treatment with ribonuclease and 

 replated on them by treatment with magnesium ribonucleate. Thus 

 Gram-positive organisms can be artificially converted to Gram- 

 negative ones and then restored to their Gram-positive state. 



In addition to this fact, it is also true that many bacteria are 

 neither definitely positive nor negative; some organisms are Gram- 

 variable and may appear either negative or positive according to 

 conditions. Other organisms contain granules which resist de- 

 colorization and may cause misinterpretation. The importance of 

 taking such variations into account has been emphasized in all 

 previous editions of this Leaflet. (Also see Committee Report, 1927.) 

 Such organisms should be recorded as Gram-variable rather than 

 made to appear either positive or negative by some modification of 

 technic. To determine whether an organism belongs to this variable 

 group, it is necessary that it be stained at two or three different ages 

 by more than one procedure. If an organism changes from positive 

 to negative or vice versa during its life history, this change should be 

 recorded, with a statement as to the age of the culture when the 

 change was first observed. It is often practical to record such an 

 organism as prevailingly positive or prevailingly negative; obviously, 

 however, this cannot be done without a very considerable series of 

 determinations. Tests must therefore be made after 1 day's and 2 

 days' incubation, sometimes also in even older cultures. It must, 

 moreover, be recognized that Gram-variable organisms are not neces- 

 sarily ones that show uneven Gram staining; the latter should be 

 recorded as staining unevenly, not as Gram-variable. 



The two methods at present recommended are the ammonium ox- 

 alate method (Hucker) and KopelofI and Beerman's modification of 

 the Burke technic. In the former the manipulation is more simple; 

 but the latter is understood to give better results if the organism is 

 growing in a medium that may be of acid reaction, and is claimed to 

 distinguish better between true and false positive reactions. These 

 two procedures are given in Leaflet IV. 



